Polymerase chain reaction (PCR) amplification of portions of the genome of both rapidly growing mycobacteria and nocardiae, followed by restriction fragment length polymorphism (RFLP) analysis of the amplification products, has proven to be a useful technique in the diagnostic laboratory. Identification of many isolates to the species level can be obtained within a few days of organism isolation using this technique, as compared with the month or more required for conventional identification based on biochemical testing. In addition, these molecular procedures allow better discrimination among species and subspecies than is possible with biochemical testing, and facilitate the detection of hitherto undescribed species. Our work with two different areas of the Nocardia genome (a portion of the gene for 16S ribosomal RNA and a portion of the gene for the heat-shock protein) has resulted in our detecting clinically significant isolates belonging both to species of Nocardia that have rarely been recognized as pathogens (such as Nocardia veterana), and to species that have been hitherto undescribed. An account of our work with one such species (Nocardia kruckzakiae) is currently in press, and work is ongoing with a number of other clinical isolates that probably belong to other as yet undescribed species. We have used our procedures in a collaborative study of the capability of the MicroSeq 500 to identify Nocardia species when using an expanded data base; we found that while the majority of the isolates were correctly identified, a few isolates could not be unambiguously assigned to a particular species because of the relatively short length of the 16S rDNA gene sequenced. Also, we have collaborated with the research group of Dr. Richard J. Wallace, Jr., in an investigation of the extent of hererogeneity in the isolates in their collection that had been previously identified as belonging to the species Nocardia nova. We are also in the process of characterizing further several interesting Nocardia isolates we have found that possess several different 16S rRNA gene copies per cell. In some Nocardia nova isolates that we have analyzed, we have found two 16S rDNA gene copies, which may differ from each other in base pair composition. To date, in four different isolates, we have found four different patterns of 16S gene distribution. The fact that all four belong to the same species has been confirmed using DNA-DNA hybridization. A summary of our findings with these isolates has been submitted for publication, and work with other isolates containing multiple different 16S rDNA genes is ongoing. Much further work is needed to determine the taxonomic and physiological significance of these genetic differences within and between species, as well as to assess such possible species-specific differences in geographic distribution, pathogenicity, and antimicrobial susceptibility as may exist.